Cardiac mitochondrial preconditioning by Big Ca super(2+)-sensitive K super(+) channel opening requires superoxide radical generation

ATP-sensitive K super(+) channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca super(2+)-sensitive K super(+) channel (BK sub(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK sub(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O sub(2)-derived free radicals in modulating protection by putative mitochondrial (m)BK sub(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O sub(2) super(-).), and m[Ca super(2+)] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK sub(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)ben z imid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O sub(2) super(-)., or an antagonist of the BK sub(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O sub(2) super(-). generation, decreased m[Ca super(2+)], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK sub(Ca) channels; 2) NS effects are blocked by BK sub(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.